Use of an active principle from flax for use in a composition for activating cytochrome c

ABSTRACT

The present invention concerns the use, in a cosmetic composition, of an effective quantity of active principle originating from flax (genus  Linum ) to activate cytochrome c and to stimulate the functions of the mitochondria. The active principle originates from the hydrolysis of flax proteins and contains principally polypeptides or peptides. It can be used alone or in association with at least one other active principle. The invention further relates to a method of cosmetic treatment intended to protect the skin and the appendages from external aggressions and to combat against cutaneous ageing.

The present invention lies in the cosmetic and pharmaceutical field, andmore particularly in the field of dermatology. The present inventionconcerns the use, in a cosmetic composition, of an effective quantity ofactive principle originating from flax (genus Linum); to activatecytochrome c. Preferably, the active principle originates from thehydrolysis of flax proteins and principally contains polypeptides orpeptides. It can be used alone or in association with at least one otheractive principle. The invention likewise relates to the use of acosmetic composition to stimulate the functions of the mitochondria andto increase the cellular energy level. The invention further relates toa method of cosmetic treatment intended to protect the skin and theappendages from external aggressions and to combat against cutaneousageing.

The active principle, activator of cytochrome c, can likewise be used toprepare pharmaceutical compositions intended to prevent or combatagainst the pathologies linked to mitochondrial dysfunctions; forexample, certain neuromuscular or cardiac degenerations, type IIdiabetes or also certain pathologies of ageing.

The term “appendages” according to the invention encompasses all thekeratinic appendages present on the surface of the body, in particularhairs, eyelashes, eyebrows, nails and head hair.

The skin is a vital organ which covers the entire surface of the bodyand ensures protective, sensitive, immune, metabolic or thermoregulatoryfunctions. The skin, like the other organs, is subject to ageing. Now,one of the major mechanisms involved in the process of ageing is theaccumulation of oxidative damage in essential molecules such as themembrane lipids, the proteins, the DNA and most particularly themitochondrial DNA (mtDNA).

Oxidative damage is caused by the free radicals, chemically unstable andvery reactive species generated by the intracellular metabolism orexternal aggressions. These external aggressions can include: UVradiation, toxins, atmospheric pollutants, alimentary oxidants.

In the skin, a premature ageing is observed, occurring in the areasexposed to radiation, characterised by phenomena of alteration to themacromolecules (lipidic peroxidation, carbonylation of proteins),affecting in particular elastin, collagen or fibronectin. A progressivedecline of the mitochondrial functions with age has likewise been ableto be shown, probably linked to the accumulation of mutations on mtDNA(K. Singh, Ann. N.Y. Acad. Sci. 1019, 2004).

One of the important consequences of the accumulation of oxidativedamage is a reduction in the capacity of the cell to produce ATP(Porteous et al., Eur J Biochem 1998, 257(1):192-201). Thus, thephenomenon of cellular ageing is in relation to the oxidative damagewhich the cell undergoes, but also to the process of energy productionnecessary for the cell to survive.

The organism possesses defence mechanisms capable of trapping ortransforming the free radicals (enzymes, glutathione, vitamins A and E,coenzyme Q10, etc.). However, these antioxidant defence systems oftenprove to be insufficient with respect to the numerous stresses andexternal aggressions to which the organisms, and the skin in particular,are subjected.

In this context, the particular properties of cytochrome c appear asbeing particularly interesting:

Cytochrome c is a small soluble protein of 15 kDa which plays anessential role in mitochondrial function and in cellular survival.Cytochrome c is a highly conserved molecule in the majority ofeucaryotes; it is found in the mitochondria of plants, animals andnumerous unicellular organisms. Cytochrome c presents a proteicstructure organised around a porphyrin, constituted by four pyrrolenuclei, themselves linked to an iron atom.

The principal role of cytochrome c is to ensure the transfer ofelectrons due to the change in valency of the iron atom. Cytochrome cwhich is soluble thus transports the electrons from complex III(coenzyme QH2 cytochrome c reductase) to complex IV (cytochromeoxydase). The electrons, which are the substrate of the cytochromeoxydase, are then transferred by the enzyme to the oxygen.

The search for compounds which are able to stimulate the mitochondriaand to increase the cellular energy level so as to prevent or combatagainst the signs of cutaneous ageing or of damage caused by externalaggressions, such as UV rays, radiations, or exposures to toxins orpollutants, is an important preoccupation in medical and cosmeticsresearch. In this respect, solutions have been proposed such as theaddition of substances involved in the energy metabolism, and moreparticularly of intermediaries or cofactors of the Krebs cycle, such asfumarate, L-malate, acetyl CoA (WO 02064129) or else the treatment ofthe skin by substances capable of reducing the free radicals, such asvitamin C (US 2004/0086526) or L-ergothioneine (WO 9836748). Inaddition, extracts of lignans extracted from flax have been described asbeneficial for the skin (WO 2004/010965). However, to the knowledge ofthe applicant, no cosmetic or pharmaceutical composition has yet beendescribed comprising compounds of peptidic nature, originating fromflax, capable of activating cytochrome c.

The present invention has as its main objective the use of an effectivequantity of an active principle of peptidic nature, originating from thehydrolysis of flax proteins (genus Linum), in a cosmetic composition toactivate cytochrome c and to stimulate the mitochondria, with the aim ofprotecting the skin from external aggressions and of combating againstcutaneous ageing. The said active principle will be able to be usedalone or in association with at least one other active principle. Theinventors have in fact demonstrated a biological activity, and moreparticularly a dermatological and cosmetic activity, of an activeprinciple of peptidic nature originating from the hydrolysis of flax,capable of activating cytochrome c. It has been demonstrated inparticular that these polypeptides or these peptides, which constitutethe active principle, when they are applied on the skin, stimulate themitochrondrial functions to a considerable extent. This has beendemonstrated, inter alia, by an increase in the expression of cytochromec, an increase in the activity of the cytochrome oxydase and an increasein the synthesis of ATP. This new active principle, which is astimulator of the mitochondria and, more generally, is capable ofprotecting the skin from external aggressions, thus allows newtherapeutic and cosmetic perspectives to be opened up.

“Active principle capable of protecting the skin from externalaggressions” is understood to mean a hydrolysate of peptidic nature,originating from the hydrolysis of flax proteins (genus Linum), capableof presenting protective properties or of reducing apoptosis in cells ortissues subjected to a stress of physico-chemical or environmentalorigin.

“Active principle capable of activating cytochrome c” is understood tomean a hydrolysate of peptidic nature originating from the hydrolysis offlax proteins (genus Linum), capable of increasing the expression ofcytochrome c, either by the activation of the proteic synthesis (bydirect or indirect modulation of the genic expression of cytochrome c),or by the increase of the biological activity of cytochrome c, or byother biological processes such as the stabilisation of the proteincytochrome c or else the stabilisation of the messenger RNA transcripts.

“Active principle capable of stimulating the functions of themitochondria” is understood to mean entirely a hydrolysate of peptidicnature originating from the hydrolysis of flax proteins (genus Linum),capable of increasing the expression or the activity of the principalactive molecules present in the mitochondria, and more generally capableof increasing the principal energetic functions of the mitochondria;namely, the chain of oxidative phosphorylation and the synthesis of ATP.

“Of peptidic nature” is understood to mean a mixture of compounds,represented mainly by peptides or polypeptides.The term “peptide” designates a chain of two or more amino acids linkedto each other by peptidic links or by modified peptidic links; the term“polypeptide” designating a peptide of larger size.

The expression “biologically active” is understood to mean “whichpossesses an activity in vivo or in vitro which is characteristic of theactivity of the active principle according to the invention”.

The term “hydrolysate or originating from hydrolysis” designates anysubstance or mixture of substances, or isolated preparation, obtainedafter hydrolysis of vegetal manner.

The active principle according to the invention can be obtained byextraction of proteins of vegetal origin, followed by a controlledhydrolysis which releases biologically active peptidic fragments.

Numerous proteins found in plants are likely to contain biologicallyactive peptidic fragments in the core of their structure. The managedhydrolysis allows these peptidic fragments to be released. It ispossible, but not necessary to realize the invention, to extract eitherfirstly the proteins concerned and to then hydrolyse them, or to carryout the hydrolysis first on a crude extract and to then purify thepeptidic fragments. It is likewise possible to use certain hydrolysedextracts without purifying the peptidic fragments thereof correspondingto the biologically active peptides according to the invention, butnevertheless ascertaining the presence of the said fragments by suitableanalytical means.

To carry out the extraction, the entire plant can be used, or a specificpart of the plant (leaf, seed, etc.).

More particularly, according to the invention one of numerous plants areused of the linaceae family, of the genus Linum (lin). The genus Linumnumbers almost 200 species, growing particularly in the northernhemisphere. These are herbaceous plants with fibrous stems, with simpleleaves, with flowers having 5 petals. Preferably, according to theinvention, the cultivated species Linum usitatissimum L. is used.According to the invention, the vegetal material which is used will bethe seed and preferably the seed having had its envelope removed by adecortication step.

In a first stage, the plant is crushed by means of a plant crusher. Thepowder which is thus obtained can be subsequently “delipided” by meansof a conventional organic solvent (such as for example an alcohol,hexane or acetone).

The extraction of the proteins from the plant is then carried outaccording to the modified conventional process (Osborne, 1924); thecrushed plant material is placed in suspension in an alkaline solutioncontaining an adsorbent product of the insolublepolyvinylpolypyrrolidone (PVPP) type (0.01-20%); in fact, it has beenobserved that the subsequent operations of hydrolyses and purificationswere facilitated by this means. The concentration of substances ofphenolic type interacting with the proteins is thus reduced.

The soluble fraction is collected after centrifugation and filtrationstages, this crude solution therefore constituting a first form of theextract containing the proteins, the carbohydrates and possibly lipids.

The proteins are then precipitated, varying the ionic force byacidifying the medium, which allow the soluble components and thenucleic acids to be eliminated.

The precipitate is then washed by means of an organic solvent such as,for example, ethanol or methanol, then the solvent is evaporated bydrying under vacuum. The precipitate, which is rich in proteins, isreturned to solution in water or another solvent, and thereforeconstitutes a more purified form of the hydrolysate.

The extraction can likewise be realized in neutral or acid medium, stillin the presence of polyvinylpolypyrrolidone. After a filtration stage,the precipitation stage is then carried out by means of a conventionalprecipitation agent such as salts (sodium chloride, ammonium sulphate)or an organic solvent (alcohol, acetone). The precipitate which isobtained can be separated from the precipitation agents by dialysisafter returning to solution in water or another solvent.

The isolated proteic fraction according to the invention is thenhydrolysed in conditions arranged to generate soluble peptides andpolypeptides. Hydrolysis is defined as being a chemical reactioninvolving the cleavage of a molecule by water, this reaction being ableto be carried out in neutral, acid or basic medium. According to theinvention, the hydrolysis is realized chemically and/or advantageouslyby proteolytic enzymes. The use can therefore be cited of endoproteasesof vegetal origin (papain, bromelaine, ficin) and micro-organisms(Aspergillus, Rhizopus, Bacillus, etc.).

For the same reasons as previously, in this managed hydrolysis stage, aquantity of polyvinylpolypyrrolidone is added to the reaction medium.After filtration, the solution which is obtained constitutes the activehydrolysate. The active hydrolysate can be further purified so as toselect the molecular weights and the nature of the generated peptides.The fractionation can be carried out advantageously by ultrafiltrationand/or by a method of the chromatographic type.

Any of the more or less purified forms of the hydrolysate is thensolubilised in water or in any mixture containing water, then sterilisedby ultrafiltration.

The vegetal hydrolysate obtained according to the invention is analysedqualitatively and quantitatively for its physico-chemicalcharacteristics and its content of compounds of proteic and peptidicnature. Compounds of peptidic nature are understood to mean thefragments of proteins, the peptides and the free amino acids present inthe mixture. The peptides, amino acids and fragments of proteins aredosed according to conventional techniques which are well known to theman skilled in the art.

Thus, according to an advantageous embodiment of the invention, theactive vegetal hydrolysate has a pH comprised between 4 and 7, andpreferably between 5 and 6, a dry extract titrating between 1 and 8 g/l,and preferably between 2 and 5 g/l, its content of compounds of peptidicnature is comprised between 0.1 and 5 g/l, and preferably between 0.5and 2 g/l and its content of sugars is from 0.5 to 2.5 g/l.

According to an advantageous embodiment of the invention, the activeprinciple according to the invention is previously solubilised in one ormore cosmetically or pharmaceutically acceptable solvents,conventionally used by the man skilled in the art, such as water,glycerol, ethanol, propylene glycol, butylene glycol, dipropyleneglycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleumjelly, a vegetal oil or any mixture of these solvents.

According to another further advantageous embodiment of the invention,the active principle according to the invention is previouslysolubilised in a cosmetic or pharmaceutical vector such as liposomes, oradsorbed on powdery organic polymers, mineral supports such as talcs andbentonites, and more generally solubilised in, or fixed on, anycosmetically or pharmaceutically acceptable vector.

The composition which is able to be used according to the invention canconsist in particular of a composition for capillary care, and inparticular a shampoo, a conditioner, a setting lotion, a treatmentlotion, a hairdressing cream or gel, a restructuring lotion for thehair, a mask, etc. The cosmetic composition according to the inventioncan be used in particular in treatments implementing an applicationwhich is followed, or not followed, by a rinsing, or else in the form ofa shampoo.

It can likewise present itself in the form of a dye or mascara to beapplied by a brush or by a comb, in particular on the eyelashes, theeyebrows or the hair.

It is readily understood that the active principle according to theinvention can be used alone or else in association with at least oneother active principle, in a cosmetic composition or for the preparationof a pharmaceutical and/or dermatological composition.

The compositions according to the invention will be able to be appliedby any suitable manner, in particular orally, parenterally or externallytopically, and their formulation will be adapted by the man skilled inthe art, in particular for cosmetic or dermatological compositions.Advantageously, the compositions according to the invention are intendedfor an administration in a topical cutaneous manner. These compositionsmust therefore contain a cosmetically and/or dermatologically acceptablemedium, i.e. compatible with the skin and the appendages, and cover allthe cosmetic or dermatological forms. In particular, these compositionswill be able to be in the form of creams, oil-in-water emulsions, orwater-in-oil or multiple emulsions, solutions, suspensions, gels, milks,lotions, sticks or else powders, suited to an application on the skin,the lips and/or the appendages.

These compositions comprise the necessary excipients for theirformulation, such as solvents, thickeners, thinners, surfactants,antioxidants, colouring agents, preservatives, perfumes.

Advantageously, the compositions which are able to be used according tothe invention further contain other active principles intended topromote its action. Among these other active principles, the activeprinciples can be named which have an anti-radical or antioxidantaction, selected from vitamin C, vitamin E, the coenzyme Q10 andpolyphenolic plant extracts.

“Anti-radical active principles” are understood to mean any compoundcapable of trapping the free radicals. These active principles arecapable of blocking the chain reactions of the free radicals before thefinal degradation stages of the biological constituents of the skin andthus have an antioxidant activity. Among these other active principles,one can likewise name the active principles stimulating the syntheses ofthe dermic macromolecules (laminin, fibronectin, collagen), for examplecollagen peptide sold under the name “Collaxyl®” by the companyVincience.

Among these other active principles, one can finally name the activeprinciples stimulating the energetic metabolism, like the activeprinciple sold under the name “GP4G®” by the company Vincience.

According to another aspect, the composition according to the inventioncan be a sun composition, i.e. a composition assisting in the protectionagainst solar radiation. Thus, actives assisting in solar protection,such as for example, solar filters, can be advantageously added to thecomposition according to the invention.

It is readily evident that the invention is addressed to mammals ingeneral and more particularly to human beings.

The effective quantity of active principle corresponds to the quantitynecessary so as to obtain the result which is sought, namely: toactivate the cytochrome c, to stimulate the mitochondria and to increasethe cellular energy level, and more generally to protect the skin andthe appendages from external aggressions and to combat against cutaneousageing.

According to an advantageous embodiment of the invention, the activeprinciple is present in the compositions of the invention at aconcentration comprised between 0.0001% to 20% approximately, andpreferably at a concentration comprised between 0.05% and 5%approximately with respect to the total weight of the final composition.

These compositions will be able to present themselves, in particular, inthe form of an aqueous, hydroalcoholic or oily solution; anoil-in-water, water-in-oil emulsion or multiple emulsions; they can alsopresent themselves in the form of creams, suspensions, or else powders,suited to an application on the skin, the mucosa, the lips and/or theappendages. These compositions can be more or less fluid and can havethe appearance of a cream, a lotion, a milk, a serum, an ointment, agel, a paste or a foam. They can also present themselves in solid form,as a stick or can be applied on the skin in the form of an aerosol. Theycan be used as a care product and/or as a make-up product for the skin.

These compositions further comprise any additive commonly used in theenvisaged field of application and also the adjuvants necessary fortheir formulation, such as solvents, thickeners, thinners, antioxidants,colouring agents, solar filters, suntan simulating agents, pigments,charges, preservatives, perfumes, odour absorbers, cosmetic orpharmaceutical actives, essential oils, vitamins, essential fatty acids,surfactants, film-forming polymers, etc.

In all cases, the man skilled in the art will ensure that the adjuvantsand their proportions are selected in such a way as to not beprejudicial to the sought advantageous properties of the compositionaccording to the invention. These adjuvants can, for example, correspondto 0.01 to 20% of the total weight of the composition. When thecomposition of the invention is an emulsion, the fatty phase canrepresent from 5 to 80% by weight and preferably from 5 to 50% by weightwith respect to the total weight of the composition. The emulsifiers andco-emulsifiers used in the composition will be selected from thoseconventionally used in the field concerned. For example, they can beused in a proportion from 0.3 to 30% by weight, with respect to thetotal weight of the composition.

By its particular activities, the active principle according to theinvention will be able to be used advantageously in a cosmeticcomposition or for the preparation of a pharmaceutical composition.

In particular, the active principle according to the invention will beable to be used advantageously in a cosmetic preparation intended tocombat in a preventive and/or curative manner against the manifestationsof cutaneous ageing and, more specifically, so as to combat againstand/or to prevent photo-induced ageing (photo-ageing). Cutaneousmanifestations of ageing are understood to mean all modifications of theexterior appearance of the skin and the appendages due to ageing, suchas, for example, wrinkles and fine lines, withered skin, flabby skin,thinned skin, the lack of elasticity and/or of tonus of the skin, dullskin without brightness, or pigmentation blemishes of the skin,discolouration of the hair or blemishes on the nails, but likewise anyinternal modification of the skin which is not ultimately resultingsystematically in a modified exterior appearance, such as for exampleany internal degradation of the skin following an exposure toultraviolet radiation (UV). The active principle according to theinvention, or the composition containing it, will allow one to combat,in particular, against the loss of elasticity and of firmness of theskin.

The active principle according to the invention allows the skin and theappendages to be protected against all types of external aggressions.The use of the active principle, or of a composition containing it, willallow the skin and the appendages to be protected and to better resistenvironmental stresses.

The expression “external aggression” is understood to mean theaggressions which can be produced by the environment. By way of example,one can name aggressions such as pollution, UV rays, or else products ofan irritant character such as surfactants, preservatives or perfumes.Pollution is understood to mean both “external” pollution, due forexample to diesel particles, ozone or heavy metals, and “interior”pollution which can be due in particular to the emissions of solvents ofpaints, glues or wallpapers (such as toluene, styrene, xylene orbenzaldehyde), or else cigarette smoke.

The active principle according to the invention can be advantageouslyused in a cosmetic composition or for the preparation of apharmaceutical composition, as a photo-protective agent and, moreparticularly, as a photo-protective agent which is designated“secondary”. A distinction is in fact made between primaryphoto-protective agents and secondary photo-protective agents. Theprimary photo-protective agents are substances which exert a physicalpower: they are able to absorb UV radiation and to restore it in theform of heat so as to protect the skin. The secondary photo-protectiveagents are substances which generally have a biological effect; theyare, for example, agents capable of limiting the damage caused to theDNA and to the membranes by the penetration of UV radiation on the skin.

The invention further relates to use in a cosmetic composition, or forthe preparation of a pharmaceutical composition, of an effectivequantity of active principle according to the invention, the activeprinciple or the composition containing it, being intended to increasethe intracellular ATP synthesis of the cells of the skin.

The invention likewise has as an object the use in a cosmeticcomposition, or for the preparation of a pharmaceutical composition, ofan effective quantity of active principle according to the invention,the active principle, or the composition containing it, being intendedto prevent damage caused to the skin by an exposure to the sun or anexposure to ionising radiation during radiotherapies.

The invention likewise has as an object the use in a cosmeticcomposition, or for the preparation of a pharmaceutical composition, ofan effective quantity of active principle according to the invention,the active principle, or the composition containing it, being intendedto stimulate the mitochondria, in particular on the areas of the bodywhich are exposed to UV radiation.

The invention further relates to the use in a cosmetic composition, orfor the preparation of a pharmaceutical composition, of an effectivequantity of active principle as previously described, the activeprinciple, or the composition containing it, being intended to protectthe skin from damage caused by free radicals.

The invention further consists in the use of an effective quantity ofactive principle to prepare a pharmaceutical composition intended toprevent or to combat against certain pathologies linked to mitochondrialdysfunctions, for example certain neuromuscular or cardiacdegenerations, type II diabetes, certain pathologies of ageing.

The invention further consists of a method of cosmetic treatmentintended to stimulate the defences and to protect the skin and theappendages from external aggressions and to combat against cutaneousageing, characterized by the application on the skin or the appendageswhich are to be treated of a composition containing an effectivequantity of active principle according to the invention.

The invention further consists of a method of cosmetic treatmentintended to give a healthier appearance to the skin and to improve thebrightness and radiance of the complexion, characterized by theapplication on the skin which is to be treated of a compositioncontaining an effective quantity of active principle according to theinvention.

Particular embodiments of this method of cosmetic treatment likewiseresult from the preceding description. Other advantages andcharacteristics of the invention will be better apparent from readingexamples, given by way of illustration and not restrictively.

EXAMPLE 1 Preparation of Active Principle from Flax (Linum usitatissimumL.)

The active principle is obtained from plants of the species Linumusitatissimum L. Of course, the extract can be prepared from plants fromat least any one of the numerous varieties and species belonging to thegenus Linum.

In a first stage, 1 kg of decorticated flax seeds are crushed in acereal crusher. The flour which is obtained is delipided by the actionof an organic solvent, hexane. After filtration and drying under vacuum,the powder which is obtained is placed in suspension in an alkalineaqueous solution (dilution at 1/10) pH 10 containing 1% ofpolyvinylpolypyrrolidone (Polyclar V ISP). This mixture is kept understirring for a sufficiently long time to allow the solubilisation of thesoluble fractions. The extraction temperature is variable (comprisedbetween 4 and 80° C.); preferably the operation will be carried outcold. After this extraction phase, the medium is clarified bycentrifuging, then filtered on a plate filter. This filtrate whichcontains the soluble fractions of the flax is then subjected to aprecipitation of the proteins, varying the ionic force in neutral oracid medium, which allows the soluble glucidic components to beeliminated, the lipids and the nucleic acids, the medium is brought topH 3.5. The supernatant is eliminated and the precipitate is then washedby means of a solvent such as, for example, ethanol or methanol then thesolvent is evaporated by drying under vacuum.

At this stage, one obtains approximately 50 grams of powder of lightyellow colour of crude bruteic extract containing

-   -   Proteins: 75%    -   Carbohydrates: 20%    -   Lipids: 5%

The precipitate, rich in proteins, is returned to solution in water oranother solvent.

The crude proteic extract is then subjected to a series of managed andselective hydrolyses consisting of chemical and enzymatic hydrolyses inthe presence of 0.5% of PVPP (Polyclar V) and of cysteine endopeptidases(papain, ficin). After reaction, the hydrolysate is filtered on a platethen on sterilising cartridge (0.2 μm).

A light-coloured hydrolysate is then obtained, titrating from 15 to 30g/l of dry extract, which is then diluted such that the concentration ofcompounds of peptidic nature determined by the Lowry method, iscomprised between 0.1 and 5 g/l and preferably between 0.5 and 2 g/l.The physico-chemical analysis of the vegetal hydrolysate, whichconstitutes the active principle, shows that its pH is comprised between4 and 7, and preferably between 5 and 6, the dry extract titrates from 1to 8 g/l and preferably between 2 and 5 g/l, its content of compounds ofpeptidic nature is comprised between 0.1 and 5 g/l, and preferablybetween 0.5 to 2 g/l and its content of sugars between 0.5 to 2.5 g/l.

EXAMPLE 2 Preparation of Active Principle from Flax (Linum usitatissimumL.)

A variant of the protocol of Example 1 consists in carrying out the samesequence of managed and selective enzymatic hydrolyses, but in thepresence of 0.5% of PVPP.

One then obtains a light-coloured hydrolysate titrating from 15 to 30g/l of dry extract after sterilising filtration.

One then proceeds to an ultrafiltration of the solution on a MilliporeHelicon filtration cartridge (cutoff threshold; 1 kDa). The highmolecular weights contained in the retentate are eliminated, thefiltrate is retained.

The concentration of compounds of peptidic nature is determined by theLowry method, either comprised between 0.1 and 5 g/l and preferablybetween 0.5 and 2 g/l. The physico-chemical analysis of the vegetalhydrolysate, which constitutes the active principle, shows that its pHis comprised between 4 and 7, and preferably between 5 and 6, the dryextract titrates between 1 to 8 g/l, and preferably between 2 and 5 g/l,its content of compounds of peptidic nature is comprised between 0.1 and5 g/l and preferably between 0.5 to 2 g/l and its content of sugarsbetween 0.5 to 2.5 g/l.

Another variant consists in carrying out a purification of the activeprinciple, obtained according to Example 1 or 2, by ion exchangechromatography, on a gel TSK column (TosoHaas) with a pH7 phosphatebuffer.

EXAMPLE 3 Demonstration of the Stimulating Effect of the ActivePrinciple According to Example 1 on the Synthesis of Intracellular ATP

The aim of this study is to determine the influence of the activeprinciple according to Example 1 on the synthesis of ATP, produced bythe mitochondria.

Protocol: This study is carried out by means of a kit “ATPBioluminescence Assay Kit HS II” (Roche Applied Science). Dermalfibroblasts are treated with a 1% solution of active principle accordingto Example 1, for a period of from 1 to 3 hours. At the end of theincubation, the wells are rinsed with 2 ml of cold PBS before adding 250μl of a lysis buffer provided by the kit. The cells of each well arethen scraped, then collected in 14 ml tubes. Each well is rinsed with2×500 μl cold PBS and the whole is collected again in the respectivetubes. From these samples, a dilution is carried out at 1/12000^(th) incold PBS before each reading. The ATP dosage is carried out on thesesamples: 50 μL of this dilution are deposited in a luma cuvette and 50μL of luminol are added. After 10 seconds, the reading of theluminescence is started. The values are standardised with respect to thequantity of proteins for each sample. The measurements are carried outby means of an apparatus: the Biocounter M2010A LUMAC®/3M.

Results: The dosages of ATP show that there is a great increase in thequantity of intracellular ATP after 1 hour and 3 hours, in cells treatedby the active principle according to Example 1, in comparison with thenon-treated cells.

Conclusion: The active principle according to Example 1 increasesappreciably the energetic level of cutaneous cells such as fibroblasts,and more generally stimulates the mitochondrial functions.

EXAMPLE 4 Demonstration of the Activating Effect of the Active PrincipleAccording to Example 1 on the Expression of Cytochrome c

The aim of this study is to determine the influence of the activeprinciple according to Example 1 on the expression of cytochrome c. Forthis, the quantity of cytochrome c was evaluated by the technique ofimmuno-transfer (or Western blot).

Protocol: Human dermal fibroblasts are treated with a 1% solution ofactive principle according to Example 1, for 72 hours. The cells arethen lysed and homogenised by sonication, then centrifuged for 10minutes at 1000 g. The samples, which are standardised for their proteinconcentration (dosage kit BCA, Pierce), are subjected to anelectrophoresis on gel Bis-TRIS 4-12% (InvitroGen). Afterelectro-transfer, the membranes are incubated for one night with ananti-cytochrome c antibody, diluted at 1/500 (Mouse monoclonal anticytochrome c, TEBU). A secondary antibody, coupled to peroxydase anddiluted at 1/5000 is then used (peroxydase conjugated F(ab′)₂, Goatantimouse, Immunotech). The chemiluminescent signal is then quantifiedwith the aid of the kit Supersignal West Femto Trial kit and read in areading chamber (Multilmage light Cabinet, Alpha ImmunotechCorporation).

Results: A clear increase is observed of the expression of cytochrome cin the fibroblasts treated by the active principle according to Example1.

Conclusions: The active principle according to Example 1 greatlystimulates the expression of cytochrome c in the cutaneous cells, andmore generally the mitochondrial functions.

EXAMPLE 5 Demonstration of the Activating Effect of the Active PrincipleAccording to Example 1 on the Enzymatic Activity of the CytochromeOxydase

The aim of this study is to determine the influence of the activeprinciple according to Example 1 on the mitochondrial activity. Forthis, the total enzymatic (mitochondrial) activity of the cytochromeoxydase was measured.

Protocol: Normal human fibroblasts are treated with a 1% solution ofactive principle according to Example 1 for 3 hours and 24 hours. Thecells are collected, rinsed, then lysed by sonication. A firstcentrifuging is carried out to eliminate the principal cellular debrisand the supernatant is then centrifuged again at 10 000 g for 10 min.The enzymatic activity is dosed in the supernatant and/or in the 2^(nd)cell pellet by a biochemical method, with the aid of the kit Cytocox 1(Sigma), then standardised for the content of proteins (dosed by the kitPCA, Pierce).

Results: The dosages show that there is a very great increase in theenzymatic activity of the cytochrome oxydase after 3 hours and 24 hoursof application of the active principle according to Example 1, incomparison with the non-treated cells.

Conclusion: The active principle according to Example 1 stimulates verygreatly the enzymatic activity of the cytochrome oxydase, and moregenerally the mitochondrial activity, in the cutaneous cells.

EXAMPLE 6 Demonstration of the Protective Effect of the Active PrincipleAccording to Example 1 on the Mitochondrial Membrane Potential

The aim of this study is to determine the protective effect of theactive principle according to Example 1, with respect to mitochondria ofdermal fibroblasts subjected to an oxidative stress, caused byoxygenated water (H₂O₂) or a UVB irradiation. For this, one uses amarker of the membrane potential of the mitochondria (JC-1). JC-1 is amarker which emits a different fluorescence according to the level ofpolarisation of the mitochondrial membrane.

Protocol: The human dermal fibroblasts are treated with a 1% solution ofactive principle according to Example 1 for 96 hours, then subjected toan oxidative stress caused by H₂O₂ at 2 mM for 30 minutes, or to anirradiation by UVB at 50 mJ/cm². Controls which are not treated by thepeptide or by H₂O₂ or are not irradiated are realized under the sameconditions. At the end of the experiment, the cells are washed, fixedand subjected to a marking by a solution of JC-1 (Molecular Probes) at0.2 μg/ml, so as to reveal the membrane potential of the mitochondria.

Results: In the fibroblasts treated by the active principle according toExample 1, the mitochondria present a red fluorescence (JC-1 aggregate),the sign of a high membrane potential, greater than in the controlcells. The fibroblasts which were irradiated or subjected to anoxidative stress have mitochondria which fluoresce little in the redrange, and principally in the green range (monomeric JC-1), the sign ofan alteration of the mitochondrial membrane potential. Under theselatter conditions, the application of the active principle according toExample 1 allows a more marked red fluorescence to be observed.

Conclusions: The application of the active principle according toExample 1 causes an increase in the membrane potential of themitochondria. Moreover, the active principle according to Example 1efficiently protects the mitochondria of the cutaneous cells subjectedto an oxidative stress or to an irradiation by UVB.

EXAMPLE 7 Preparation of Compositions

1—Sun protection cream:

Commercial names INCI names % mass PHASE A Demineralised water Aqua(Water) qs Pemulen TR1 Acrylates/C10-30 Alkyl Acrylate 0.40 CrosspolymerGlycerine Glycerin 3.00 Nipastat Sodium Sodium Methylparaben (and)Sodium 0.15 Ethylparaben (and) Sodium Butyl paraben (and) SodiumPropylparaben (and) Sodium Isobutylparaben PHASE B Parsol MCX EthylhexylMethoxycinnamate 7.50 Eusolex 4360 Benzophenone-3 3.00 Parsol 1789 ButylMethoxydibenzoylmethane 2.00 Myritol 318 Caprylic/Capric Triglyceride4.00 Emulgade SEV Hydrogenated Palm Glycerides (and) 5.00 Ceteareth-20(and) Ceteareth-12 (and) Cetearyl Alcohol Propylparaben Propylparaben0.15 Nacol 16-98 Cetyl Alcohol 1.00 PHASE C TEA Triethanolamine 0.20PHASE D Active principle of 3 Example 1 Perfume Parfum (Fragrance) qsColouring agent qs

The constituents of Phase A and Phase B are heated separately between70° C. and 75° C. Phase B is emulsified in Phase A under stirring. PhaseC is added, at 45° C., increasing the stirring. Phase D is then addedwhen the temperature is below 40° C. The cooling is continued to 25° C.under strong stirring.

2—After-sun milk:

Commercial names INCI names % mass PHASE A Montanov L C14-22 Alcohols(and) C12-20 3.00 Alkyl Glucoside Waglinol 2559 Cetearyl Isononanoate4.00 Tegosoft TN C12-15 Alkyl Benzoate 3.00 Apricot kernel oil PrunusArmeniaca (Apricot) Kernel 2.00 Oil Avocado oil Persea Gratissima(Avocado) Oil 1.00 Abil 350 Dimethicone 1.00 PHASE B Demineralised waterAqua (Water) qs PHASE C Simulgel EG Sodium Acrylate/Acryloyldimethyl 0.4Taurate Copolymer (and) Isohexadecane (and) Polysorbate 80 Copolymer(and) Polysorbate 80 PHASE D Phenonip Phenoxyethanol (and) 0.30Methylparaben (and) Ethylparaben (and) Butylparaben (and) Propylparaben(and) Isobutylparaben Ethylparaben and Propylparaben and ButhylparabenGermall 115 Imidazolidinyl Urea 0.20 PHASE E Active principle of 0.1Example 1

Prepare Phase A under stirring. Incorporate the xanthan gumprogressively, under deflocculating stirring. Phases C and D will beincorporated once the gel is finished. Phase E, prepared previously upto perfect dissolving of the DHA, will then be added. Adjust the pH ifnecessary to 4-4.5. Colour and perfume.

3—Anti-ageing cream:

Commercial % names INCI names mass Phase A Montanov 68 Cetearyl Alcohol(and) Cetearyl Glucoside 6.00 Squalane Squalane 3.00 Cetiol SB 45Butyrospermum Parkii (Shea Butter) 2.00 Waglinol 250 CetearylEthylhexanoate 3.00 Amerchol L-101 Mineral Oil (and) Lanolin Alcohol2.00 Abil 350 Dimethicone 1.50 BHT BHT 0.01 Coenzyme Q10 Ubiquinone 0.10Phase B Avocado oil Persea Gratissima (Avocado) Oil 1.25 PhenonipPhenoxyethanol (and) Methylparaben (and) 0.75 Ethylparaben (and)Butylparaben (and) Propylparaben (and) Isobutylparaben Phase CDemineralised water Aqua (Water) qs Butylene Glycol Butylene Glycol 2.00Glucam E10 Methyl Gluceth-10 1.00 Allantoin Allantoin 0.15 CarbopolUltrez 10 Carbomer 0.20 Phase D TEA Triethanolamine 0.18 Phase E Activeprinciple of 0.5 Example 1 GP4G Water (and) Artemia Extract 1.50Collaxyl Water (and) Butylene Glycol (and) 3.00 Hexapeptide-9 Phase FPerfume Parfum (Fragrance) qs Colouring agent qs

Prepare and melt Phase A at 65-70° C. Heat Phase C at 65-70° C. Phase Bis added to Phase A just before emulsifying A in B. At approximately 45°C., the carbomer is neutralised by addition of Phase D. Phase E is thenadded under light stirring and the cooling is continued to 25° C. PhaseF is then added, if desired.

4—Protective day cream:

Commercial names INCI names % mass Phase A Emulium Delta Cetyl alcohol(and) Glyceryl Stearate 4.00 (and) PEG-75 Stearate (and) Ceteth-20 (and)Steareth-20 Lanette O Cetearyl Alcohol 1.50 D C 200 Fluid/ Dimethicone1.00 100 cs DUB 810C Coco Caprylate/Caprate 1.00 DPPG Propylene GlycolDipelargonate 3.00 DUB DPHCC Dipentaerythrityl Hexacaprylate/ 1.50Hexacaprate Cegesoft PS6 Vegetable Oil 1.00 Vitamin E Tocopherol 0.30Phenonip Phenoxyethanol (and) Methylparaben 0.70 (and) Ethylparaben(and) Butylparaben (and) Propylparaben (and) Isobutylparaben Phase BDemineralised water Aqua qsp 100 Glycerine Glycerin 2.00 Carbopol EDT2020 Acrylates/C10-30Alkyl Acrylate 0.15 Crosspolymer Keltrol BT XanthanGum 0.30 Phase C Sodium Hydroxide Sodium Hydroxide 0.30 (sol. 10%) PhaseD Demineralised water Aqua 5.00 Stay-C 50 Sodium Ascorbyl Phosphate 0.50Phase E Butylene Glycol Butylene Glycol 2.00 Dekaben CP Chlorphenesin0.20 Phase F GP4G Water (and) Artemia Extract 1.00 Active principle of 5Example 1

Prepare Phase A and heat at 75° C. under stirring. Prepare Phase B,dispersing the carbopol, then the xanthan gum under stirring. Allow torest. Heat at 75° C.

At temperature, emulsify A in B under rotor-stator stirring. Neutralisewith Phase C under rapid stirring. After cooling at 40° C., add Phase D,then Phase E. The cooling is continued under light stirring and Phase Fis added.

1-15. (canceled)
 16. Flax seed hydrolysate (genus Linum), wherein, said active principle originates from the hydrolysis of flax proteins, said active principle is of peptidic nature, pH of said active principle is comprised between 4 and 7, dry extract of said active principle is titrates between 1 and 8 g/l, content of compounds of peptidic nature of said active principle is comprised between 0.1 and 5 g/l, and content of sugars of said active principle is comprised between 0.5 and 2.5 g/l.
 17. Flax seed hydrolysate according to claim 16, wherein, said active principle originates from flax seeds of the species Linum usitatissimum L.
 18. Flax seed hydrolysate (genus Linum) according to claim 16, wherein said active principle is previously solubilised in one or more cosmetically or pharmaceutically acceptable solvents, selected among water, glycerol, ethanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylated or propoxylated diglycols, cyclic polyols, petroleum jelly, a vegetal oil or any mixture of these solvents.
 19. A cosmetic composition comprising as an active principle, a flax seed hydrolysate (genus Linum) according to claim 16, used alone or in association with at least one other active principle, in a cosmetically or dermatologically acceptable medium.
 20. The composition according to claim 19 wherein the active principle is used in a quantity representing from 0.0001% to 20% of the total weight of the composition
 21. The composition according to claim 20, wherein the active principle is used in a quantity representing from 0.05% to 5% of the total weight of the composition.
 22. The composition according to claim 19, wherein the said composition is in a form suited to application in a topical manner.
 23. The composition according to claim 19, further comprising at least one other active principle promoting the action of the said active principle.
 24. The composition according to claim 23, wherein the other active principle is selected from the antioxidant active principles and/or the active principles stimulating the synthesis of the extracellular matrix, and/or the active principles stimulating the energetic cellular metabolism.
 25. A method of preparing a cosmetic composition activating cytochrome c, stimulating the mitochondria and increasing the synthesis of intracellular ATP of the cells of the skin, comprising adding an effective quantity of an active principle originating from the hydrolysis of flax proteins (genus Linum) in a cosmetically or dermatologically acceptable medium.
 26. A method of activating cytochrome c, stimulating the mitochondria and increasing the synthesis of intracellular ATP of the cells of the skin, comprising topically applying of an effective quantity of an active principle of peptidic nature, wherein, said active principle is in a cosmetic composition, and said active principle is originating from the hydrolysis of flax proteins (genus Linum)
 27. The method according to claim 26, wherein, said cosmetic composition is intended to protect the skin and the appendages against all types of external aggression.
 28. The method according to claim 27, wherein, said external aggressions are UV radiations.
 29. The method according to claim 26, wherein, said cosmetic composition is intended to prevent or to treat the cutaneous signs of ageing and/or of photo-ageing.
 30. The method according to claim 26, wherein, said cosmetic composition is intended to improve the brightness and the radiance of the complexion.
 31. A method of preventing or combating against mitochondrial dysfunctions, such as certain neuromuscular or cardiac degenerations, type II diabetes or certain pathologies of ageing, comprising administering to a subject in need an effective quantity of active principle originating from the hydrolysis of flax proteins (genus Linum) in a pharmaceutical composition. 